The mechanism by which bone cell networks respond to load-induced mechanical signals is poorly understood. The long term goals of this project are to gain a better understanding of mechanotransduction in bone cell networks, composed of multiple cell types, and how disrupting this mechanotransduction in vivo affects load-induced osteogenesis. We propose that gap junctional intercellular communication and release of nucleotides, specifically adenosine triphosphate (ATP), from osteocytic or osteoblastic cells, are both essential to maximize bone cell response to the physical environment. Our central hypothesis is that biophysical signals, such as fluid flow, stimulate osteoblast proliferation and differentiation via a mechanism involving mobilization of cytosolic Ca2+, activation of GJIC and release of ATP through gap junction hemichannels. We will examine this hypothesis through the completion of four specific aims: 1) quantify the effect of fluid flow, in the presence and absence of agents that inhibit cytosolic Ca2+ mobilization, on GJIC, activation of GJ hemichannels and release of ATP by bone cells;2) examine the effect of fluid flow on bone cell proliferation;3) examine the effect of fluid flow on bone cell differentiation and 4) examine load-induced osteogenesis in bones isolated from connexin deficient mice. During this five-year project we will utilize a novel co-culture fluid flow apparatus, shRNA strategies, site-directed mutagenesis an innovative proteomics approach, a well characterized in vivo bone loading apparatus and transgenic murine models to examine whether osteocytic cells exposed to mechanical signals communicate proliferation and differentiation inducing signals to osteoblastic cells, a dogma of bone cell biology with surprisingly little experimental support, and if so the importance of this to in vivo mechanotransduction. An understanding of how mechanical signals are detected by bone cells and communicated throughout the bone cell network is important to understanding how bone adapts to its physical environment. This will in turn provide insights as to novel therapeutic targets for many musculoskeletal pathologies. Additionally, an understanding of how mechanical signals regulate bone cell proliferation and differentiation would be beneficial in designing in vitro environments, i.e. bioreactors, for novel bone tissue engineering protocols.